Within recent years, the art has recognized the potential commercial significance of enzymes possessing specificity in hydrolyzing alpha-1,6-glucosidic starch linkages. Conventional amylases are not completely effective in hydrolyzing 1,6 starch linkages and frequently form undesirable starch hydrolyzate by-products. By employing alpha-1,6-glucosidic hydrolyzing enzymes (e.g., pullulanase) in conjunction with other amylases, the conversion syrup industry would be able to produce a broader spectrum of starch hydrolyzates and conversion syrup products. If pullulanase were available at an economically feasible cost, starch conversion syrups of a superior quality could be produced at a significantly lower price.
In general, pullulanase is produced by inoculating and fermenting a culture media containing assimilable carbon, nitrogen and mineral nutrients with Aerobacter aerogenes under conditions conductive to its growth. In some processes, pullulanase production by the Aerobacter aerogenes is permitted to occur simultaneously with cell propagation. In other processes, pullulanase elaboration does not occur until late in growth phase. In some processes, pullulanase production is intentionally repressed by employing a culture media deficient in saccharide materials which are deemed essential for pullulanase synthesis. Upon achieving sufficient Aerobacter aerogenes growth, the cells are then induced to produce pullulanase with a carbohydrate such as maltose, maltotriose or pullulan.
As a class, the Aerobacter aerogenes strains heretofore employed in the pullulanase production stage generally possesses a characteristic of producing divergent levels of extracellular enzyme, tightly bound enzyme and superficially bound enzyme when different carbohydrate source materials are employed. The ratio of extracellular to cell bound enzyme produced by an Aerobacter aerogenes organism is often reversed simply by altering or substituting one carbohydrate for another in the fermentation process. Depending upon the particular carbohydrate source, the fermentation process will normally favor either extracellular pullulanase production (e.g., more than 75%) or cell bound pullulanase production (e.g., about 75% or more).
As a class, Aerobacter aerogenes organisms are incapable of elaborating pullulanase when dextrose is employed as a sole carbohydrate source. However, if the propagation and pullulanase production is conducted simultaneously in the presence of an excess amount of a carbohydrate inducer, extracellular pullulanase is primarily elaborated (e.g., about 75-80%) with the remaining portion being tightly bound on the organisms. When equivalent weights of dextrose (for growth) and carbohydrate inducer (e.g., maltose) are employed for cell propagation and enzyme production, extracellular pullulanase production will usually decrease (e.g., about 20%) while superficially cell bound pullulanase increases to a level of about 70% with the remaining portion of the total enzyme being in a tightly bound form. By conducting the Aerobacter aerogenes pullulanase process via a cell propagation and subsequent pullulanase production by addition of inducers thereto, the Aerobacter aerogenes will primarily produce cell bound enzymes at a level of about 75% with the balance being extracellular enzyme.
In Biochemische Zietschrift 334, 79-95 (1961) H. Bender and K. Wallenfels reported that Aerobacter aerogenes belonging to the Enterobactericeae family, produced pullulanase. Inducers such as maltose, maltotriose or pullulan were reported as being an essential carbohydrate for pullulanase production. Pursuant to this process, about 75-80% of the total pullulanase elaborated by the organisms is extracellular. Due to the difficulties in purifying and recovering the pullulanase, Wallenfels et al..sup.1 subsequently proposed propagating the organism in the presence of equal amounts of glucose and maltose. By this method extracellular pullulanase was reportedly decreased to a level of about 20% or less, with the remaining pullulanase being localized near the organisms surface. This localized pullulanase was disclosed as being easily liberated by surfactants. FNT 1 -- Biochemical & Biophysical Research Communications, Vol. 22, No. 3, 1966 -- pp. 254-261.
In U.S. Pat. Nos. 3,654,087, 3,654,088 and 3,654,089 (by Coker et al.) it is disclosed that improved pullulanase yields are achieved when Aerobacter aerogenes pullulanase production is conducted in a two-stage process. In the first stage of the Coker et al. process, Aerobacter aerogenes growth is favored without appreciable pullulanase production. This is generally accomplished by conducting the fermentation in the presence of carbohydrates such as dextrose as a sole or principle carbohydrate. Upon achieving a high level of cell propagation, the Aerobacter aerogenes are then induced to produce pullulanase with carbohydrate inducers. In U.S. Pat. No. 3,654,088, it is reported that improved pullulanase yields are achieved in the production stage when induced cells are incubated in the presence of amylopectin. In general, the pullulanase produced by the Aerobacter aerogenes in the aforementioned Coker et al. patents is primarily cell bound enzyme (e.g., about 65-75% or more).
Numerous patents have reported different microbial strains as being capable of producing .alpha.-D-1,6-glucosidic hydrolyzing enzymes (e.g., see U.S. Pat. Nos. 3,560,345 by Yokobayashi et al. Pseudomonas amyloderamosa, 3,716,455 by Ueda et al. Escherichia intermedia and British Pat. No. 1,260,418.
Pullulanase production with culture mediums containing starch hydrolyzates have also been reported. In U.S. Pat. No. 3,560,345 by Yokobayashi et al., any carbohydrate material having .alpha.-1,4 or 1,6-glucosidic linkages are reported as a suitable carbon source for the synthesis of extracellular isoamylases. According to the Yokobayashi et al. patentees, extracellular yields of about 180-220 units/ml..sup.2 are reported via incubation in culture mediums containing maltose or soluble starch as a sole carbohydrate source. Table 5 of the Yokobayashi et al. patent indicates that pullulanase yields from the incubation of a culture media comprised of maltose, ammonium salts and soybean hydrolyzates (up to 130 units/ml) are greater than those wherein starch hydrolyzates are employed as a carbohydrate source material. U.S. Pat. No. 3,622,460 by Masuda et al., discloses maximum yields of about 125 alpha-1,6-glucosidase units/ml..sup.2 via employing a production culture medium which contains starch hydrolyzates (D.E. of 2-15%) in combination with soybean hydrolyzates as a nitrogen source material. In Canadian Pat. No. 901,503 by Heady, starch hydrolyzates having a D.E. of 5-40 are used to facilitate pullulanase production. FNT 2 -- Units are determined by an iodine assay which comparatively gives higher unit values than the assay method used to define the units herein.
The art has sought a process which would provide high pullulanase yields in a readily recoverable and usable form. Some processes produce relatively high pullulanase yields in a form unsuitable for recovery. Other processes produce a readily recoverable pullulanase in low yields.
Although there is a commercial need for pullulanase at a price which would economically justify its usage in starch conversion processes, pullulanase can neither be obtained at a price nor in a quantity as necessitated by the starch syrup industry. Notwithstanding this need, the art has been unable to discover and develop an organism capable of producing high pullulanase yields under process conditions wherein the pullulanase is readily and economically recoverable in a form suitable for commercial usage.